International journal of clinical pharmacology and therapeutics
Ferulic acid alleviates Aβ25-35- and lipopolysaccharide-induced
PC12 cellular damage: a potential role in Alzheimer’s disease by
PDE inhibition
Hao Huang1,2, Zeng-Chun Ma2, Yu-Guang Wang2, Qian Hong3, Hong-Ling Tan2,
Cheng-Rong Xiao2, Qian-De Liang2, Xiang-Lin Tang2, Yue Gao2
1
College of Life Science and Bioengineering, Beijing University of Technology ,
Beijing, 100124, China;
2
Beijing Institute of Radiation Medicine, Tai-Ping Road 27, Beijing, 100850,
P.R.China;
3
No. 97 Hospital of CPLA, Xuzhou 221004, China.
Correspondence to Yue Gao, Zeng-Cun Ma, Beijing Institute of Radiation Medicine,
27 Tai-Ping Road, Beijing, 100850, PR China
[email protected]; [email protected]
Abstract Objective: Phosphodiesterase (PDE) plays an important role in the
pathogenesis of Alzheimer’s disease (AD). Ferulic acid (FA) has a therapeutic benefit
in the treatment of AD. We investigated whether this therapeutic effect is based on the
modulation of the PDE/cyclic adenosine monophosphate (cAMP) pathway. In the
present study, we investigated whether FA could abrogate Aβ25–35- and
lipopolysaccharide-induced cellular damage. Materials and Methods: Cell viability,
superoxide production, and the levels of inflammatory factors were investigated. We
further investigated the intracellular levels of cAMP and Ca2+, both of which are
associated with PDE activity. Furthermore, molecular docking was used to identify
the binding mode between phosphodiesterase 4B2 (PDE4B2) and FA. Results:
Pretreatment with FA significantly maintained cell viability, increased the levels of
superoxide dismutase, and inhibited production of TNF-α and IL-1β induced by Aβ25–
35. Moreover, pretreatment with FA increased the intracellular levels of cAMP and
decreased the intracellular levels of Ca2+. The docking results also showed that FA
has the potential to inhibit PDE4B2 activity. Conclusions: Taken together, our results
suggested that one of the therapeutic effects of FA on AD was potentially mediated by
modulating the PDE/cAMP pathway.
Key words Ferulic acid-Alzheimer’s disease-phosphodiesterase-lipopolysaccharide
Introduction
Progressive degeneration of β-amyloid peptide (Aβ) in the brain has been
proposed as a pivotal event in the pathogenesis of Alzheimer’s disease (AD), which is
associated with excessive accumulation of Aβ in the hippocampal and cerebral
cortical region. Sufficient evidence has shown that oxidative stress contributes to AD
neuropathology [1]. Antioxidants, anti-inflammatory drugs, or putative mitochondrial
protectors have been used for the treatment of AD [1, 2]. However, no effective
treatment has emerged from these efforts so far, due to toxicity and a questionable
effect in preclinical and clinical trials.
Ferulic acid (FA) is a component found in various grains, fruits, and vegetables
and is considered an effective therapeutic tool for a variety of diseases including
neurodegenerative disorders, cardiovascular disease, inflammatory diseases, and even
AD. FA can cross the blood–brain barrier easily and shows a lot of biological
activities including anti-inflammatory and anti-oxidant effects [3, 4]. Studies have
shown that FA has a neuroprotective effect against oxidative stress [5, 6]. Long-term
oral FA administration showed protective activities in β-amyloid precursor
protein/presenilin 1 (APP/PS1) transgenic mice, including a decrease in amyloid
deposition in the brain [7]. Furthermore, FA ethyl ester was reported to destabilize
preformed Aβ fibrils in vitro [8, 9]. Apart from protecting cells from peroxidative
damage as a free radical scavenger, FA also possesses another important biological
function as it might be able to increase intracellular cyclic nucleotides by inhibiting
PDE [10]. The enhancement of cyclic adenosine monophosphate (cAMP) and cAMP
response element binding protein (CREB) phosphorylation induced by FA is probably
one of the most important mechanisms in the treatment of depression and AD [11].
However, its biological effects on the central nervous system (CNS) remain largely
unknown. Our hypothesis is that FA might inhibit the PDE/cAMP pathway.
Understanding the mechanisms by which FA ameliorates AD neuropathology as a
multi-targeted compound could open new avenues for the development of innovative
treatments for AD associated with inflammation.
Material and methods
Cell culture and treatments
PC12 cells (Cell resource center, IBMS, CAMS/PUMC) were routinely cultured
in RPMI 1640 medium (Gibico, China) supplemented with 10% heat-inactivated fetal
bovine serum (Gibico, New Zealand), 5% heat-inactivated horse serum (Gibico, New
Zealand), 100 U/mL penicillin, and 100 U/mL streptomycin (HyClone, USA) at 37 °C
in a humidified atmosphere of 95% air and 5% CO2. PC12 cells (1 x 105 cells/mL) in
RPMI 1640 medium supplemented with low serum content (2% fetal bovine serum)
were seeded in a 6-well plate or a 96-well plate. The cells were allowed to grow for
24 hours before processing for further experiments.
Aggregation of Aβ25-35 and LPS preparation
Different articles have reported different times for Aβ25–35 incubation, from 0~7
days. In order to get aggregated Aβ, we incubated it at 37 oC for 6 hours, 12 hours, 24
hours, 3 days, 7 days, and 14 days, respectively. There was no obvious difference
between the cytostatic effects observed for the different incubation times. Most
researchers used a 4-day or a 7-day period for Aβ25–35 incubation [12, 13, 14]. Aβ25–35
(Sigma, USA), which is the most toxic peptide fragment derived from amyloid
precursor protein, was prepared in a stock solution of 1mM in distilled water and
aggregated by incubation at 37 °C for 7 days before use. Then the solutions were
diluted to the required concentration with serum-free RPMI 1640 medium. LPS (Sigma,
USA) was dissolved in sterile, pyrogen-free water and diluted with sterilized
phosphate-buffered saline.
Assay of cell viability
Cell proliferation and viability was measured by Cell Counting Kit-8 detection kit
(CCK-8, Dojindo Kumamoto, Japan). First, we examined the cytotoxicity of FA
(NICPBP, Beiing), Aβ25-35, and LPS. PC12 cells were treated with different
concentrations of FA (2.5~40 µM) and Aβ25-35 (2.5, 5, 10, 20, 40 µM) for 24 hours and
48 hours, respectively. Cells were treated with various concentrations of LPS (0.125,
0.15, 0.175, 0.2, 0.225 mg/mL) for 24 hours. Second, to detect the protective effect of
FA on Aβ25-35 and LPS-induced cellular damage, PC12 cells were pretreated with
various concentrations of FA (0, 2.5, 5.0,10, 20, 40 µM) for 24 hours followed by
exposure to 20 µM Aβ25-35 for 48 hours or exposure to LPS (0.15 mg/mL) for 24
hours. CCK-8 solution was applied at 10 μL per well, followed by 2 hours of
incubation at 37 °C. The absorbance values of all wells were then determined at 450 nm
in a VICTOR™ X5 Multilabel Plate Reader. The experiments were independently
repeated three times.
Superoxide assay
Superoxide dismutase (SOD) activity was measured using 96-well plates and then
using the Superoxide Dismutase Assay Kit (JianCheng, Nanjing) with some
modifications. The assay was performed according to the manufacturer’s instructions.
In brief, cell cultures grown in 96-plates were exposed to Aβ25-35 or vehicle in 150 µL
of treatment medium. 50 µL of treatment medium with and without 800 U/mL SOD
was added. Then, the supernatant was incubated at 37 °C for 20 minutes. Afterwards,
the absorbance was read at 450 nm with a VICTOR™ X5 Multilabel Plate Reader.
Cytokine assays
PC12 cells were seeded in a 96-well plate at a density of 5×105 cells/mL and then
pretreated with FA (2.5, 5, 10, 20, or 40 μM) for 24 hours followed by Aβ 25-35 (20 µM)
treatment for 48 hours. The TNF-α and IL-1β levels in the cultured supernatant were
quantified using ELISA kit (Multiscience, china) in accordance with the
manufacturer's instructions. The absorbance at 450 nm was determined using a
VICTOR™ X5 Multilabel Plate Reader.
cAMP immunoassay
PC12 cells (5x105/mL) were seeded in 12-well plates coated with poly-L-lysine
(Solarbio, China) in normal medium for 24 hours, then the cells were incubated in 1%
horse serum and 0.5% FBS for 24 hours prior to the 24-hour FA treatment and
followed by exposure to LPS (150 ng/mL) for 24 hours. Cells were treated with 0.1 M
HCl after removing the culture media and incubated for 10 minutes to verify cell lysis.
The cell lysates were centrifuged and the supernatant was used in the assay.
Measurement of total intracellular cAMP was performed using a cAMP enzyme
immunoassay kit (Jiancheng, Nanjing). The values were normalized by protein
quantification. Rolipram (the inhibitor of PDE4) was used at 30 µM which is a
concentration that can completely inhibit PDE4 activity [15, 16, 17].
Measurement of Ca2+
The intracellular calcium concentration was measured by using fluorescent dye
Fura-2/AM (Sigma, USA). First, we observed the typical plots of changes in
intracellular Ca2+ levels over time after the addition of different FA concentrations (5,
10, or 20 µM). Second, we investigated the effect of FA on LPS-induced Ca2+ influx.
PC12 cells were pretreated with various concentrations of FA (0, 2.5, 5.0,10, 20, 40
µM) for 24 hours followed by exposure to LPS (0.15 mg/mL) for another 24 hours.
After the above treatment, PC12 cells were loaded with 2 μg/mL Fura- 2/AM dye in
the presence of pluronic acid at 37 ºC for 40 minutes in HEPES-buffered solution.
After incubation, the cells were gently washed three times with HBSS (Kaixinjie,
Beijing). Images were acquired on a PerkinElmer UltraVIEW VoX Confocal Imaging
System. Changes in the fluorescence ratio were measured at an emission wavelength of
510 nm for a dual excitation wavelength of 340 and 380 nm. The changes in
fluorescence were recorded for at least 5 minutes.
Docking
We downloaded the crystal structure of PDE4B2 from the RCSB Protein Data
Bank (PDB ID: 1R06) [18]. The visual tool Pymol was used to analyze the
construction of the binding site of PDE4B2. The file of FA was obtained from
PubChem Compound Database. The PDB formats were transformed to PDBQT
formats by MGLTools 15.2. Molecular docking was conducted by AutoDock 4.0
software to analyze the conformation of FA and the PDE4B2.
Results
Protective effects of FA on Aβ25-35-induced cytostatics in PC12 cells
When PC12 cells were treated with 2.5 µM to 500 µM FA for 24 hours. 2.5~80
µM FA increased cell viability (Figure 1 A), and the viability of PC12 cells could also
be increased within the range of 2.5~80 µM FA. Therefore, to demonstrate the real
effect of FA on PC12 cells, an Aβ25-35 concentration of 2.5~40 µM was chosen for the
following experiments. The inhibitory effect of Aβ25-35 on cell proliferation was
detected by CCK8 assay. As shown in Figure 1B, when the PC12 cells were treated
with 0, 2.5, 5, 10, 20, and 40 µM Aβ25-35 for 48 hours, Aβ25-35 significantly inhibited
the proliferation of PC12 cells at concentrations higher than 10 µM at 48 hours. Based
on the results for the toxic effect of Aβ25-35 on PC12 cells, treatment with Aβ25-35 at a
concentration of 20 µM for 48 hours will be appropriate for our next study. To
investigate the protective effects of FA on Aβ25-35 induced neurotoxicity, PC12 cells
were pretreated with FA at concentrations of 2.5, 5, 10, 20 and 40 µM for 24 hours
and then exposed to 20 µM Aβ25-35 for 48 hours. Cell viability increased at all
concentrations of FA compared to the Aβ25-35-treated group (Figure 1C).
FA promotes generation of SOD and inhibits generation of TNF-α and
IL-1β
To further explore whether the neuroprotective effect of FA results from its
antioxidant effect, the supernatant of the PC12 cells culture medium was collected for
SOD assay. The results showed that with the application of Aβ25-35 alone, the activity
of SOD decreased significantly compared to the control group, but under pretreatment
with FA, the activity level of SOD significantly increased compared to the Aβ25-35
group (Figure 2A). As shown in Figure 2 (B, C), Aβ25-35 markedly increased the
release of pro-inflammatory cytokines TNF-α and IL-1β into culture supernatants of
PC12 cells compared with the control group. FA significantly attenuated the release of
TNF-α and IL-1β into the medium compared with the Aβ25-35 treated group, although
lower concentration of FA (2.5 µM) did not significantly affect the release of these
inflammatory mediators.
Protective effect of FA on cellular damage induced by LPS
LPS (0.125, 0.15, 0.175, 0.2, 0.225 mg/mL) induced cell death in a
dose-dependent manner (Figure 3A). Cell viability decreased to approximately 75% at
a concentration of 0.15 mg/mL over 24-hour incubation. FA significantly protected
PC12 cells from the toxic effect of LPS when the cells were preincubated with FA for
24 hours prior to LPS exposure. All concentrations (ranging from 2.5 to 40 µM)
caused significant increases in viability as compared with negative controls (LPS
alone) (Figure 3B).
Effect of FA on cAMP level
In this study, 150 ng/mL LPS were used to stimulate cAMP production [19, 20].
There was a clear increase of cAMP levels in FA- and rolipram-treated PC12 cells as
compared with negative controls (LPS alone). FA was shown to inhibit the
LPS-induced cAMP decrease at concentrations of 10, 20, and 40 µM. It was also
observed that FA increased cAMP levels in a dose dependent manner (Figure 4). The
results showed that FA has the potential to increase the cAMP level in PC12 cells.
Effect of FA on Ca2+ level
The intracellular Ca2+ level was stable throughout the recording periods in PC12 cells
of the control group. A concentration-dependent increase in the intracellular Ca2+
levels was displayed in PC12 cells exposed to different concentrations of FA (Figure
5A-D). As shown in Figure 5E, compared with the control group, the fluorescence
intensity of Fura-2/AM in PC12 cells increased evidently after incubation with LPS
alone, while pretreatment with FA or rolipram obviously attenuated the increase of
Ca2+ induced by LPS. The results suggested that the neuroprotective effect of FA was
strongly linked with reduction of the incremental Ca2+ influx.
Results of docking
The predicted binding sites and the molecular docking results are shown in Figure 6A,
B. The conformation with the lowest binding energy (－6.36k cal·mol-1) was regarded
as the optimal conformation. The conformations of FA are located in the hydrophobic
cavity area, which is composed by amino acid residues including Tyr233, His234,
Met347, Asn395, Phe414, Gln443, and Phe446. Abundant irregular coiling structures
and helix structures can be found in the hydrophobic cavity area.
Discussion
Kanski J et al. suggested that 10~50 µM FA exerts protective effects against
oxidative stress-mediated changes by hydroxyl and peroxyl radical generators in
protein oxidation, lipid peroxidation, and ROS. FA could potentially be of importance
for the treatment of AD and other diseases related with oxidative stress [21]. In this
study, we found that FA has no cytotoxic effects in a wide range of concentrations
(0.5~500 µM) and could increase PC12 cell viability within concentrations of 2.5~80
µM. Pretreatment with FA for 24 hours reverses the inhibition of cell viability induced
by Aβ25-35 and LPS. It has also been reported that FA is better for nerve cell
proliferation than brain-derived neurotrophic factor (BDNF) in the prevention and
treatment of some degenerative retinal diseases [22]. FA promotes viability of
Schwann cells and may be useful in the development of future strategies for the
treatment of peripheral nerve injury [23].
While different cell lines react differently to FA treatment, FA significantly
inhibited both viability and activation of HSC-T6 cells in vitro. Thus, FA has
antifibrotic potential in renal and cardiac disease [24]. Altogether, literature shows
that FA has versatile biological functions.
There is considerable evidence that the neuronal damage caused by Aβ is
mediated by damage to membranes caused by free radicals. Oxidative stress is
involved in various neurodegenerative diseases, including Alzheimer’s disease and
Parkinson’s disease, and is defined as an impaired balance between the production of
reactive oxygen species (ROS) and antioxidant defense [25]. In the brain of
Alzheimer’s disease patients, oxidative stress is evident. Antioxidant enzymes such as
superoxide dismutase (SOD) play a key role in diminishing oxidative stress, which is
considered to be a therapeutic approach for treatment of various neurological diseases
[26]. Our study also showed that the application of FA significantly increased the
activity levels of SOD in PC12 cells compared to the group treated with Aβ25-35;
therefore, FA should have a significant nerve protection potential.
Furthermore, we observed that pretreatment with FA significantly decreased the
release of proinflammatory cytokines TNF-α and IL-1β into culture supernatants of
PC12 cells in a concentration-dependent manner induced by Aβ25-35. Our results are
consistent with previous reports that FA has a protective effect against
proinflammatory responses. Cytokines play an essential role in the organization and
regulation of inflammatory responses [27]. Neuroinflammation is an important
pathoetiologic hallmark of AD [2, 28]. FA ameliorated neuroinflammation and
decreased expression of proinflammatory cytokines (TNF-α and IL-1β) in PSAPP
mice [5]. However, it remains possible that FA may have an anti-inflammatory effect
as a multi-targeted compound. In order to further confirm our hypothesis that FA has
the potential effect of treating AD by inhibiting the PDE/cAMP pathway, LPS was
used to induce PC12 cellular damage and decrease cAMP levels in PC12 cells. FA
was shown to inhibit the LPS-induced cellular damage and increased cAMP levels in
PC12 cells. It was also observed that FA increased cAMP levels in a dose-dependent
manner.
There has been substantial evidence that repeated LPS treatment clearly induces
an up-regulation of PDE4A, PDE4B, and PDE4D subtypes and a significant
down-regulation of cAMP/pCREB/BDNF signaling pathway in the hippocampus and
prefrontal cortex of mice [29]. cAMP is thought to be the main intracellular second
messenger and to play a crucial role in regulating inflammation; furthermore, cAMP
has been recognized as a regulator of innate immunity and reactive oxygen species
[30]. Specific PDE inhibitors have been shown to improve memory performance in
different animal models of AD. The specific PDE4 inhibitor rolipram was found to
effectively restore cognitive deficits in animal models of AD, which shows that it
modulates the activity of cAMP-mediated signaling and regulates CREB
phosphorylation. The PDE3 inhibitor etazolate was effective in preventing the
depressive-like behavior induced by LPS treatment in mice. Etazolate alleviates
depressive-like behavior by up-regulating cAMP, pCREB, and BDNF levels in the
prefrontal cortex and hippocampus [29]. More recently, PDE5 inhibitors have also
been shown to effectively restore memory function by elevating the levels of cyclic
guanosine monophosphate (cGMP) [31]. Yabe T et al. found that FA increased cAMP
response element binding protein (CREB) phosphorylation and ameliorated the
stress-induced depression-like behavior of mice[32]. Examination of PDE4B mRNA
of PC12 cells revealed a decrease in PDE4B, while immunoblotting showed
up-regulation of CREB and phospho-CREB with FA pretreatment. These results are
consistent with the increased levels of cAMP. More detailed results will be released in
the near future (data are not published).
It has been reported that LPS induced Ca2+ overload in PC12 cells. In this study,
we examined the prohibiting effects of FA on intracellular Ca2+ accumulation induced
by LPS. FA has shown the effect of decreasing the intracellular Ca2+ induced by LPS
treatment. Extracellular fluid at sites of injury and inflammation has been reported to
contain high concentrations of Ca2+, which also suggests a possible role for
extracellular Ca2+ as a danger signal. LPS treatment induced Ca2+ influx into neurons,
promoted a transient elevation of intracellular calcium, and led to the activation of
Ca2+-mediated signal transduction [33]. In cultured hippocampal cells, glutamate
toxicity significantly increased the intracellular Ca2+ concentration, whereas this
increase in Ca2+ level was inhibited by FA treatment. It was also reported that sodium
ferulate significantly attenuates anoxia and reoxygenation-induced Ca2+ overload and
improves cell survival [3].
The cross-talk between intracellular Ca2+ and cyclic nucleotide levels existed in
various types of cells. PDE4 is one of the major PDE isoforms present in vascular
endothelial cells. Studies indicated that the effects of rolipram (the inhibitor of PDE4)
and the cAMP analogue dibutyryl cAMP (db-cAMP) are similar to those of Gingko
biloba extract EGb 761 on agonist-induced Ca2+ increases. This effect of inhibiting
PDE4 activity likely involves an elevation of cAMP levels and a subsequent
modification of calcium signaling in endothelial cells [34]. Of course, further study is
needed to clarify the role of FA on Ca2+ influx.
Molecular docking results have shown that FA interacts strongly with critical
amino acid residues including Tyr233, His234, Met347, Asn395, Phe414, Gln443,
and Phe446 at the FA-binding site of PDE4B2. Furthermore, these amino acid
residues maybe very important for inhibitor binding.
Based on the above results, it is an open question whether FA is only activating
adenylate cylase activity alone or in combination with the inhibition of PDE, thereby
increasing the intracellular level of cAMP and then to activate PKA. To examine the
effects of FA on the LPS-inducible PDE expression, PDE activity and resultant
cellular cAMP levels are vital to deeply understand the mechanism of its anti-AD
effect. Further investigation is needed to address the relationship between FA,
inflammatory factors, and PDE at gene and protein levels, and also behavioral tests in
the studied animals should be included as this may provide more experimental support
for the treatment of AD.
Conclusion
Our present study showed that FA inhibits Aβ25-35- and LPS-induced neurotoxicity.
This may be mediated by inhibition of PDE/cAMP signaling pathway. These results
suggested possible therapeutic uses of FA for the treatment of AD, where cAMP and
Ca2+ mediated neuroinflammation play a significant role.
Acknowledgements
This work was supported by funding from the National Natural Science
Foundation of China (No. 81130067 and No. 81202936).
Conflict of interest
The authors report no conflicts of interest. The authors alone are responsible for
the content and writing of the paper.
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Figure 1.
Protective effects of FA on Aβ25-35-induced cytostatics in PC12 cells.
(A) Effects of FA on PC12 cell proliferation. PC12 cells were treated with different
concentrations of FA for 24 hours. Cell viability was measured with CCK8 assay.
(B) Toxic effect of Aβ25-35 on PC12 cells. Cells were plated and treated with Aβ25-35
for 48 hours. Cell viability was measured with CCK8 assay. The results are expressed
as percentage of the absorbance of control cells. (C) Protective effects of FA on
Aβ25-35-induced PC12 cellular damage. PC12 cells were preincubated with the
indicated concentrations of FA for 24 hours, then exposed to Aβ25-35 (20 uM) and
cultured for 48 hours. Results are indicated as means ± SD of three experiments.
*p<0.05 vs vehicle group, #p<0.05 vs Aβ25-35 group.
Figure 2. FA promotes generation of SOD and inhibits generation of TNF-α and
IL-1β. PC12 cells were preincubated for 24 hours with the indicated concentrations of
FA, then exposed to Aβ25-35 (20 µM) and cultured for 48 hours. The levels of SOD
(A), TNF-α (B), and IL-1β (C) in the culture supernatants were determined. Aβ25-35
significantly reduced SOD activities compared to the control group. Pretreatment with
FA produced a great increase in SOD activities compared to Aβ25-35 alone. The levels
of TNF-α and IL-1β were measured using ELISA kits. Results are means ± SD of
three experiments. *p<0.05 vs vehicle group, #p<0.05 vs Aβ25-35 group.
Figure 3. Protective effects of FA on LPS-induced cytotoxicity in PC12 cells. (A)
Cells were treated with various concentrations of LPS for 24 hours, CCK8 assay was
used to determine cell viability. LPS induced cell death in a dose-dependent manner.
(B) A significant increase in cell viability was observed when cells were pretreated
with various concentrations of FA for 24 hours prior to LPS exposure. Results are
means ± SD of three experiments. *p<0.05 vs vehicle group, #p<0.05 vs Aβ25-35
group.
Figure 4. Effect of FA on cAMP level. FA prevents the down-regulation of cAMP
levels in the PC12 cells induced by LPS. PC12 cells were preincubated with the
indicated concentrations of FA for 24 hours, then exposed to LPS (150 ng/mL) for 24
hours. The levels of cAMP were measured using ELISA kits. LPS significantly
reduced cAMP levels as compared to the control group. Results are means ± SD of
three experiments. *p<0.05 vs vehicle group, #p<0.05 vs LPS group.
Figure 5. FA decreases intracellular Ca2+ levels. (A) The intracellular Ca2+ of PC12
cells in the control group was stable during the observation time. (B), (C), (D):
Representative plots of changes in intracellular Ca2+ levels over time after the addition
of different FA concentrations (5, 10 or 20 µM). (E) PC12 cells were pretreated with
FA (2.5, 5, 10, 20, 40 µM) for 24 hours, followed by LPS (0.15 mg/mL) treatment for
24 hours. The enhancement of intracellular Ca2+ induced by LPS was retarded in
PC12 cells with FA pretreatment. Results are means ± SD of three experiments.
*p<0.05 vs vehicle group, #p<0.05 vs LPS group.
Figure 6. Molecular docking results. (A) Predicted binding sites in PDE4B2. (B)
Close view of binding mode of FA with PDE4B2 active site residues. Hydrogen
bonds are represented by yellow dotted lines.
Fig. 1 (A)
Fig. 1 (B)
Fig. 1 (C)
Fig. 2 (A)
Fig. 2 (B)
Fig. 2 (C)
Fig. 3 (A)
Fig. 3 (B)
Fig. 4
Fig. 5 (A), (B), (C), (D), (E)
Fig. 6 (A)
Fig.6 (B)